Substituted n-acyl homoserine lactones

ABSTRACT

The substituted N-acyl homoserine lactones have the formula (I) wherein R is a saturated or unsaturated straight chain or branched chain aliphatic hydrocarbyl group containing from 5 to 14 carbon atoms; R 2  is H or a  1 - 4 C alkyl group; R 3  is H or F; and any enantiomer thereof. These compounds exhibit immunosuppressant activity while exhibiting reduced biosensor (autoinducer) activity compared to known N-acyl homoserine lactones.

The invention relates to substituted N-acyl homoserine lactones. Moreparticularly, it relates to certain substituted N-acyl homoserinelactones which exhibit immunosuppressant activity while exhibitingreduced biosensor (autoinducer) activity. The invention, further,relates to a pharmaceutical composition containing such a substitutedN-acyl homoserine lactone as an active ingredient.

Immunosuppressant compounds induce an inhibition of the immune responsesystem. Compounds which are known to exhibit immunosuppressant activityinclude the fungal metabolite Cyclosporin A and the macrolide antibiotic(a metabolite from Streptomyces tsukabaensis) termed FK506. Both ofthese agents have been used clinically and experimentally to suppressthe immune system in transplantation and in the treatment of a number ofdiseases.

Autoimmune diseases are disorders where the host discrimination of“self” versus “non-self” breaks down and the individual's immune system(both acquired and innate components) attacks self tissues. Thesediseases range from common entities, such as rheumatoid arthritis,thyroid autoimmune disease and type 1 diabetes mellitus to less commonentities, such as multiple sclerosis and to rarer disorders such asmyasthenia gravis. Advances in basic biomedical science and, inparticular, in immunology have indicated that the main and fundamentallesion responsible for the induction and persistence of most autoimmunediseases resides within auto-reactive proliferating T lymphocytes. Infact, the majority of autoimmune diseases are linked to a loss of T cellhomeostasis. The healthy immune system is held in balanced equilibrium,apparently by the contra-suppressive production of cytokines by T helper1 (Th1) and T helper 2 (Th2) lymphocyte subsets. In autoimmunity, Th1cytokines predominate; in allergy, Th2 cytokines take their place. Acytokine intimately associated with the development of Th1 biasedresponses and, consequently, autoimmune disease is TNF-α.

Both Cyclosporin A and FK506 have been used clinically in the treatmentof autoimmune diseases with encouraging results.

The currently available immunosuppressant drugs have the disadvantage ofa narrow therapeutic index, i.e. toxicity versus clinical benefit. Thecompounds are known to be nephrotoxic, neurotoxic and potentiallydiabetogenic and this has limited their use in the fields mentionedabove. Problems also exist with the administration of these compounds,their bioavailability and the monitoring of their levels both clinicallyand in the laboratory.

N-Acyl homoserine lactones are known. WO 92/18614 discloses compoundshaving the formula

where n is 2 or 3; Y is O, S or NH; X is O, S or NH and R is anoptionally substituted C₁-C₁₂ alkyl or acyl. These compounds weredisclosed in that document as autoinducers and as agents for the controlof gene expression. A naturally occurring autoinducer is a compoundproduced by a microorganism during metabolism which then acts toincrease the expression of the genes of the microorganism. Compounds inthe same series as disclosed in WO 92/18614 are also mentioned inJournal of Bacteriology, volume 175, number 12, June 1993, pages 3856 to3862 but again there is no teaching that they might have any effectoutside microorganisms.

G. Papaccio, Diabetes Res. Clin. Pract. vol. 13, No. 1, 1991, pages95-102 discloses the use of N-acetylhomocysteine thiolactone as anenhancer of superoxide dismutase in an attempt to increase protectionagainst chemically induced diabetes.

The use of N-acetylhomocysteine thiolactone to modify the IgE moleculeis taught by J. Ljaljevic et al in Od. Med. Nauka, vol. 24, 1971, pages137-143 and Chemical Abstracts, vol. 78, No. 7, February 1973, abstractNo. 41213a. However, there is no suggestion in this paper ofimmunosuppression.

U.S. Pat. No. 5,591,872 discloses the compound N-(3-oxododecanoyl)homoserine lactone as the autoinducer molecule for Pseudomonasaeruginosa. In “Infection and Immunity”, vol. 66, No. 1, January 1998,the authors report the action of N-(3-oxododecanoyl) homoserine lactone(OdDHL) in inhibiting the concavalin A mitogen stimulated proliferationof murine spleen cells and TNF-α production by LPS-stimulated adherentmurine peritoneal macrophages.

In WO 95/01175, a class of compounds related to those compoundspreviously disclosed in WO 92/18614 was described as exhibitingantiallergic activity and inhibiting histamine release.

A new subclass of immunosuppressant N-acyl homoserine lactones wasdisclosed in WO 01/74801. These compounds, which may be represented bythe formula

in which R is an acyl group of the formula

wherein one of R¹ and R² is H and the other is selected from OR⁴, SR⁴and NHR⁴, wherein R⁴ is H or 1-6C alkyl, or R¹ and R² together with thecarbon atom to which they are joined to form a keto group, and R³ is astraight or branched chain, saturated or unsaturated aliphatichydrocarbyl group containing from 8 to 11 carbon atoms and is optionallysubstituted by one or more substituent groups selected from halogen,1-6C alkoxy, carboxy, 1-6C alkoxycarbonyl, carbamoyl optionally mono- ordisubstituted at the N atom by 1-6C alkyl and NR⁵R⁶ wherein each of R⁵and R⁶ is selected from H and 1-6C alkyl or R⁵ and R⁶ together with theN atom form a morpholino or piperazino group, or any enantiomer thereof,are reported as being capable of modulating the immune response in theliving animal body. In particular, they have an inhibitory effect onlymphocyte proliferation in humans and downregulate TNF-α secretion bymonocytes/macrophages and, in consequence, the activation of Th1lymphocytes in humans. All of these N-acyl homoserine lactones disclosedin the prior art have biosensor activity which is a disadvantage in acompound also possessing anti-proliferative activity. It has been theaim of research workers in this field to find compounds related to OdDHLwhich have comparable immune modulation properties but which have theadvantage that they exhibit reduced biosensor activity. It is to thecredit of the inventors that such compounds have now been discovered.

Accordingly, the present invention in a first aspect provides a compoundof the formula I

in which R¹ is a saturated or unsaturated, straight chain or branchedchain aliphatic hydrocarbyl group containing from 5 to 14 carbon atoms;R² is H, or a 1 to 4C alkyl group; R³ is H or F, and any enantiomerthereof.

The group R¹ in the formula I above is a saturated or unsaturated,straight chain or branched chain aliphatic hydrocarbyl group containingfrom 5 to 14 carbon atoms. For example, R¹ may be a straight or branchedchain 5 to 14C alkyl group or a straight or branched chain 5 to 14Calkenyl or 5 to 14C alkynyl group. Preferably, R¹ is a straight chainalkyl group containing from 5 to 14 carbon atoms. Specific examples ofsuch alkyl groups include pentyl, hexyl, heptyl, octyl, nonyl and decylgroups. According to a particularly preferred embodiment of theinvention, the group R¹ is n-octyl.

The group R² in the formula I above is selected from the groupcomprising H and 1 to 4C alkyl. Examples of 1 to 4C alkyl groups thatmay be represented by R² in the formula I above include methyl, ethyl,propyl, isopropyl, n-butyl and isobutyl groups. Preferably R² in thepresent invention is either H or methyl.

The group R³ in the formula I above is selected from H and fluoro. Itwill be apparent that when R³ in the formula I is a fluoro group, anadditional chiral centre will be present at the carbon atom to which thefluoro group is attached. The present invention relates to individualenantiomeric forms of the compounds as well as the racemic mixtures.

The preferred compounds according to the present invention have theformula I above in which the group R¹ is n-octyl, the group R² is H ormethyl and the group R³ is H or fluoro. Specific examples of compoundsthat are preferred according to the invention areN-(4-aza-3-oxododecanoyl)-L-homoserine lactone,N-(4-methyl-4-aza-3-oxododecanoyl)-L-homoserine lactone andN-(4-aza-2-fluoro-3-oxododecanoyl)-L-homoserine lactone.

The compounds of the present invention can, in general, be prepared bythe reaction of a substituted carboxylic acid having the formulaR¹R²NC(O)CHR³C(O)OH, where R¹, R² and R³ are defined above, withN,N′-carbonyldiimidazole and then reacting the N-acylimidazoleintermediate with L-homoserine lactone, typically as the hydrochloride.

The carboxylic acids described above as starting materials in thepreparation may, themselves, be obtained by the reaction of the amineR¹R²NH with an ethyl ester of the optionally-substituted malonic acid toform the ethyl ester of the required substituted carboxylic acid whichmay then be hydrolysed to the carboxylic acid. For instance, we havefound that substituted carboxylic acids of the formula above, wherein R³is H, can be prepared simply by the reaction of the amine R¹R²NH withethyl malonyl chloride and then hydrolysing the resulting ethyl ester tothe acid. In the case where the group R³ is a fluoro group, we havefound that we can obtain the ethyl ester of the desired substitutedcarboxylic acid in good yield from the reaction between the amine R¹R²NHand dimethyl fluoromalonate.

The compounds of the present invention have use as pharmaceuticallyactive ingredients in the treatment of an individual suffering from adisease or disorder which is responsive to the activity of animmunosuppressant. The compounds of the present invention inhibit theproliferation of lymphocytes and the production of TNF-α while, at thesame time, exhibiting reduced autoinducer activity compared to OdDHL.Thus, the compounds may be used to modulate the immune system in anindividual, for instance in the treatment of autoimmune disease.Specific examples of autoimmune disease that may be treated by theadministration of an effective dose of a compound of the inventioninclude psoriasis, multiple sclerosis and rheumatoid arthritis. A dosageto be administered to an individual in need of therapy will, of course,depend on the actual active compound used, the mode of administrationand the type of treatment desired as well as on the body mass of theindividual. The active compound may be administered on its own or in theform of an appropriate medicinal composition containing, for instance,an appropriate pharmaceutical carrier, excipient or diluent. Othersubstances can also be used in such medicinal compositions, such asantioxidants and stabilisers, the use of which are well known to personsskilled in the art.

EXAMPLES Example 1 Synthesis of N-(4-aza-3-oxododecanoyl)-L-homoserinelactone (4-aza-OdDHL) Step 1. Ethyl 4-aza-3-oxododecanoate

To an ice-cold stirred solution of n-octylamine (101.54 mmol, 13.12 g)in dry dichloromethane (75 ml) was added ethyl malonyl chloride (50.77mmol, 6.5 ml) dropwise over the period of 15 minutes and further stirredfor one hour. The solution was sequentially washed with saturated sodiumbicarbonate (3×20 ml), 2 M HCl (3×20 ml) and saturated sodium chloridesolution (1×20 ml). Drying over MgSO₄ and rotary evaporation of organicsolvent gave ethyl 4-aza-3-oxododecanoate as white solid (8.5 g, 69%).

TLC R_(f) 0.43, Ethyl acetate-hexane (1:1)

¹H NMR (250 MHz, CDCl₃) δ 0.93 (3H, t, J 6.0 Hz, (CH₃(CH₂)₇), 1.07-1.77(13H, m, CH₃(CH₂)₅ and OCH₂CH₃), 1.48 (2H, m, CH₃(CH₂)₅CH₂), 3.23 (2H,t, J 7.5 Hz, CH₃(CH₂)₆CH₂), 3.30 (2H, s, COCH₂COO), 4.23 (2H, q, J 7.5Hz, OCH₂CH₃), 7.71 (1H, br s, NH).

Step 2. 4-Aza-3-oxododecanoic acid

To a stirred solution of ethyl 4-aza-3-oxododecanoate (7.5 mmol, 1.81 g)in ethanol (50 ml) was added a solution of NaOH (8.75 mmol, 350 mg) inwater (50 ml) and stirring continued at room temperature for 16 hours.This mixture was rotary evaporated to remove ethanol. The residue wasredissolved in water and washed with ethyl acetate. It was thenacidified with 2 M HCl (pH 1). The product 4-aza-3-oxododecanoic acidwas extracted with dichloromethane (4×15 ml). After drying over MgSO₄and rotary evaporation of dichloromethane, 4-aza-3-oxododecanoic acidwas obtained as a white solid (1.35 g, 84.3%).

¹H NMR (250 MHz, CDCl₃) δ 0.9 (3H, t, J 6.15 Hz, CH₃(CH₂)₇), 1.3 (10H,m, CH₃(CH₂)₅), 1.54 (2H, m, CH₃(CH₂)₅CH₂—), 3.3 (2H, J 6.4,CH₃(CH₂)₆CH₂), 3.37 (2H, s, COCH₂CO), 7.19 (1H, s, NH), 10.64 (1H, s,COOH).

Step 3. N-(4-aza-3-oxododecanoyl)-L-homoserine lactone (4-aza-OdDHL)

To a stirred solution of 4-aza-3-oxododecanoic acid (5.74 mmol, 1.23 g)in dry dichloromethane (40 ml) was added sequentiallyN,N′-carbonyldiimidazole (5.02 mmol, 943 mg), triethylamine (5.74 mmol,0.8 ml) and L-homoserine lactone hydrochloride (5.74 mmol, 790 mg). Themixture was stirred at room temperature overnight and then solventrotary evaporated. The residue was redissolved in ethyl acetate (40 ml)and the solution washed with saturated sodium bicarbonate (2×25 ml), 2 MHCl (3×25 ml) and brine (1×25 ml). After drying over MgSO₄ and removalof solvent in vacuum, the isolated product 4-aza-OdDHL was purified byPLC using Hexane-Ethyl acetate (1:4) solvent system (676 mg, 39.5%)

TLC R_(f) 0.23, Ethyl acetate-hexane (4:1)

IR (KBr) 3293 (NH), 1771 (ring C═O), 1685 (3-amide C═O), 1645 (1-amideC═O) cm⁻¹

¹H NMR (250 MHz, CDCl₃) δ 0.9 (3H, t, J 6.5 Hz, CH₃(CH₂)₇), 1.27 (10H,m, CH₃(CH₂)₅), 1.5 (2H, br, CH₃(CH₂)₅CH₂) 2.34 (1H, dd, J 9.9 and 1.5Hz, ring, 4α-H), 2.66 (1H, ddd, J 2.0, 3.8 and 6.8 Hz, ring, 4β-H), 3.22(2H, t, J 6.6 Hz, CH₃(CH₂)₆CH₂), 3.28 (2H, s, COCH₂CO), 4.23 (1H, ddd, J3.0, 4.4 and 6.3 Hz, ring, 3H), 4.49 (1H, dd, J 7.65 and 1.3 Hz, ring,5α-H), 4.6 (1H, ddd, J 2.3, 4.9 and 8.7 Hz, ring, 5β-H), 7.14 (1H, br t,4-NH), 8.2 (1H, d, J 6.8 Hz, NH-HSL).

ES-MS m/z 299.13 (M+H, C₁₅H₂₆N₂O₄ requires m/z 298), 321.11 (M+Na).

Example 2 Synthesis of N-(4-aza-4-methyl-3-oxododecanoyl)-L-homoserinelactone

(4-aza-4-Me-OdDHL)

Step 1. Ethyl 4-aza-4-methyl-3-oxododecanoate

The procedure described above in Example 1, step 1, was repeated exceptthat N-methyl-n-octylamine was used instead of n-octylamine. The titleproduct was obtained as a semisolid that was recrystallised from diethylether (97%).

TLC R_(f) 0.5, Ethyl acetate-hexane (1:1)

¹H NMR (250 MHz, CDCl₃) δ 0.87 (3H, t, J 6.7 Hz, (CH₃(CH₂)₇), 1.26-1.32(13H, m, CH₃(CH₂)₅ and OCH₂CH₃), 1.55 (2H, s (broad), CH₃(CH₂)₅CH₂),2.94, 2.98 (3H, 2×s, NCH₃), 3.24 (1H, dd, J 1.9 and 7.6 Hz,CH₃(CH₂)₆CH(H)), 3.39 (1H, ddd, J 1.9, 5.6 and 7.6 Hz, CH₃(CH₂)₆CH(H)),3.44 (2H, s, COCH₂COO), 4.2 (2H, two q (overlap), OCH₂CH₃).

Step 2. 4-Aza-4-methyl-3-oxododecanoic acid

Ethyl 4-aza-4-methyl-3-oxododecanoate (4.85 mmol, 1.246 g) wassaponified with a solution of NaOH (5.7 mmol, 226 mg) according to theprocedure described above for ethyl 4-aza-3-oxododecanoate in Example 1,Step 2. The product 4-aza-4-methyl-3-oxododecanoic acid was obtained asa white solid (1.03 g, 93%).

¹H NMR (250 MHz, CDCl₃) δ 0.89 (3H, t, J 6.0 Hz, (CH₃(CH₂)₇), 1.3 (10H,m, CH₃(CH₂)₅), 1.58 (2H, br s, CH₃(CH₂)₅CH₂), 3.03 (3H, s, NCH₃), 3.24(2H, J 7.6 Hz, CH₃(CH₂)₆CH₂), 3.42 (2H, s, COCH₂COO), 10.15 (1H, s,COOH).

Step 3. N-(4-aza-4-methyl-3-oxododecanoyl)-L-homoserine lactone(4-aza-4-Me-OdDHL)

4-Aza-4-methyl-3-oxododecanoic acid (3.9 mmol, 896 mg) was coupled withL-homoserine lactone hydrochloride (4.0 mmol, 550 mg) according to theprocedure described in Example 1, Step 3. The title product,4-aza-4-methyl-OdDHL was purified by PLC in Hexane-Ethyl acetate (1:4),(237 mg, 19.5%).

TLC R_(f)-0.19, Ethyl acetate-hexane (4:1)

IR (KBr) 3299 (NH), 1776 (ring C═O), 1683 (3-amide C═O), 1654 (1-amideC═O) cm⁻¹

¹H NMR (250 MHz, CDCl₃) 0.88 (3H, t, J 5.6 Hz, (CH₃(CH₂)₇), 1.27 (10H,m, CH₃(CH₂)₅), 1.53 (2H, br s, CH₃(CH₂)₅CH₂), 2.29 (1H, dd, J 10.1 and1.47 Hz, ring, 4α-H), 2.66 (1H, dd, J 2.0 and 5.6 Hz, ring, 4β-H), 3.0(3H, 2×s, NCH₃), 3.3 (2H, t, J 7.72 Hz, CH₃(CH₂)₆CH₂), 3.38 (2H s,COCH₂CO), 4.28 (1H, dddd, 1.49, 3.19, 4.05 and 6.1 Hz, ring, 3H), 4.48(1H, t, J 9.1 Hz, ring, 5α-H), 4.6 (1H, dddd, J 1.5, 2.6, 4.5 and 7.2Hz, ring, 5β-H), 8.84 (1H, d, J 7.4 Hz, NH-HSL).

ES-MS m/z 313.14 (M+H, C₁₆H₂₈N₂O₄ requires m/z 312), 335.12 (M+Na).

Example 3 4-Aza-2-fluoro-3-oxododecanoyl-L-homoserine lactone(4-aza-2-F-OdDHL) Step 1. Methyl 4-aza-2-fluoro-3-oxododecanoate

To a stirred solution of dimethyl fluoromalonate (1 mmol, 150.11 mg) inanhydrous methanol (10 ml) was added a solution of n-octylamine (1 mmol,166 μl) in methanol (5 ml) drop wise over the period of one hour at roomtemperature. It was then further stirred at room temperature for twohours. The solution was rotary evaporated and the residue redissolved inethyl acetate. The ethyl acetate solution was sequentially washed with 2M HCl (2×10 ml) and saturated sodium chloride (1×15 ml) solution. Dryingover MgSO₄ and rotary evaporation of organic solvent gave white solidthat was a mixture of bisamide and ethyl4-aza-2-fluoro-3-oxododecanoate. Desired product was purified as a whitesolid by PLC in 40-60 petroleum ether-diethyl ether (3:2), (117 mg,47%).

TLC R_(f) 0.23, petroleum ether-diethyl ether (3:2)

¹H NMR (400 MHz, CDCl₃) δ 0.88 (3H, t, J 6.4 Hz, (CH₃(CH₂)₇), 1.29 (10H,m, CH₃(CH₂)₅), 1.54 (2H, br s, CH₃(CH₂)₅CH₂), 3.32 (2H, m, J 6.6 Hz,CH₃(CH₂)₆CH₂), 3.9 (3H, s, OCH₃), 5.28 (1H, d, J 49.2, C(H)F), 6.42 (1H,s, NH).

Step 2. 4-Aza-2-fluoro-3-oxododecanoic acid

To a stirred solution of methyl 4-aza-2-fluoro-3-oxododecanoate (0.28mmol, 70 mg) in methanol (15 ml) was added a solution of NaOH (0.28mmol, 11.2 mg) in water (5 ml) and further stirred at room temperaturefor two hours. This mixture was rotary evaporated to remove methanol.The residue was redissolved in water (25 ml) and aqueous solution waswashed with ethyl acetate. It was then acidified with 2 M HCl (pH 1).The product 4-aza-2-fluoro-3-oxododecanoic acid was extracted in ethylacetate (3×15 ml). After drying over MgSO₄ and rotary evaporation ofethyl acetate, 4-aza-2-fluoro-3-oxododecanoic acid was obtained as awhite solid (64 mg, 99%).

¹H NMR (400 MHz, CDCl₃) δ 0.9 (3H, t, J 6.6 Hz, (CH₃(CH₂)₇), 1.31 (10H,m, CH₃(CH₂)₅), 1.60 (2H, m, CH₃(CH₂)₅CH₂), 3.4 (2H, J 6.8 Hz,CH₃(CH₂)₆CH₂), 5.3 (1H, d, J 47.2 Hz, C(H)F), 6.7 (1H, s, NH), 7.3 (1H,br s, COOH)

Step 3. 4-Aza-2-fluoro-3-oxododecanoyl-L-homoserine lactone(4-aza-2-F-OdDHL)

To a stirred solution of 4-aza-2-fluoro-3-oxododecanoic acid (0.28 mmol,64 mg) in dry dichloromethane (20 ml) was added sequentially1,1′-carbonyldiimidazole (0.3 mmol, 49 mg), triethylamine (0.3 mmol, 42μl) and L-homoserine lactone hydrochloride (0.3 mmol, 42 mg). Thismixture was stirred at room temperature overnight and then solventrotary evaporated. The residue was redissolved in ethyl acetate (30 ml).The ethyl acetate solution was washed with saturated sodium bicarbonate(2×20 ml), 1 M KHSO₄ (2×20 ml) and brine (1×20 ml). It was dried overMgSO₄ and solvent rotary evaporated. The product 4-aza-4-methyl-OdDHLwas purified by PLC in ethyl acetate (8.2 mg, 10.4%).

TLC R_(f) 0.32, Ethyl acetate

IR (KBr) 3298 (NH), 1772 (ring C═O), 1683 (3-amide C═O), 1653 (1-amideC═O) cm⁻¹

¹H NMR (400 MHz, CDCl₃) 0.89 (3H, t, J 6.9 Hz, (CH₃(CH₂)₇), 1.3 (10H, m,CH₃(CH₂)₅), 1.56 (2H, m, CH₃(CH₂)₅CH₂), 2.27 (1H, dd, J 8.97 and 2.3 Hz,ring, 4α-H), 2.80 (1H, ddd, J 1.4, 2.54 and 5.34 Hz, ring, 4β-H), 3.33(2H, m, CH₃(CH₂)₆CH₂), 4.28 (1H, dd, J 1.4 and 4.5 Hz, ring, 3H), 4.53(1H, m, ring, 5α-H), 4.64 (1H, ddd, J 2.64, 2.94 and 4.14 Hz, ring,5β-H), 5.3 (1H, dd, J 10.2 and 38.0 Hz, CF(H)), 6.63 (1H, s, 4-NH), 7.5(1H, dd, J 13.3, 6.4 Hz, NH-HSL).

ES-MS m/z 317.12 (M+H, C₁₅H₂₅FN₂O₄ requires m/z 316), 339.09 (M+Na).

Immunomodulatory Activity of Homoserine Lactone Compounds Materials andMethods I. ConA Mitogen-Stimulated Proliferation of Murine Splenocytes

The concanavalin A (ConA) cell proliferation assay was used to assessthe effect of test homoserine lactone (HSL) compounds on T-cellactivation and proliferation. Proliferation was assessed by theincorporation of [³H]-thymidine into DNA. Eight-week-old female BALB/cmice were obtained from Harlan (Bicester, Oxon, UK) and given food andwater ad libitum. Splenocyte suspensions were prepared by removing thespleens and placing them into RPMI 1640 medium. The spleens were forcedthrough 70-μm-pore-size wire gauzes using the plunger from a 5-mlsyringe to produce a single cell suspension. The cells were pelleted bycentrifugation, and erythrocytes were lysed with 0.017 M Tris, 0.144 Mammonium chloride buffer, pH 7.2. Leucocytes were washed twice with RPMI1640 medium with 2% (vol/vol) foetal calf serum (FCS) and resuspended incomplete cell culture medium (CTCM) consisting of RPMI 1640 medium with5% FCS, 2 mM L-glutamine, and 5×10⁻⁵ M 2-mercaptoethanol. HSL compoundswere tested at doubling down dilutions ranging from 1 mM to 0.1 μM in afinal volume of 200 μl of CTCM, containing ConA (Sigma, Poole, UK) at 1μg/ml and 100,000 spleen cells. Following incubation for 48 h at 37° C.in 5% CO₂-air, 0.25 μCi[³H]-thymidine (Amersham) in 10 μl volume made upin RPMI 1640 medium was added and the cells were incubated for a further24 h. Cells were harvested onto fibreglass filters with a Packardfiltermate harvester. After the addition of 25 μl of MicroScint-O(Packard) to each well, the filters were counted with the PackardTopCount scintillation counter.

Mitogen (Concanavalin A) induced murine splenocyte proliferation wasindicated by the incorporation of tritated thymidine into the DNA in themouse spleen cells as shown by counts per minute using the scintillationcounter. The inhibitory effect of an HSL compound being tested on cellproliferation was indicated by a reduction in counts per minute.

FIG. 1 shows the plots of counts per minute (CPM) against theconcentrations (micromolar) of the prior art compound OdDHL and thecompound N-(4-aza-3-oxododecanoyl)-L-homoserine lactone (4-aza-OdDHL).As can be seen from this Figure, 4-aza-OdDHL, like OdDHL, inhibitssplenocyte proliferation. FIG. 2 shows plots similar to those in FIG. 1except, here, a comparison is made of the plot obtained for OdDHL withthat obtained for the compoundN-(4-methyl-4-aza-3-oxododecanoyl)-L-homoserine lactone(4-methyl-4-aza-OdDHL). As shown in FIG. 2, the compound4-methyl-4-aza-OdDHL inhibits splenoctye proliferation. FIGS. 1 and 2also show the plots of autoinducer activity of the HSL compounds againstHSL concentration (micromolar).

The IC₅₀ value, i.e. the dose of compound (micromolar) required toinhibit 50% of a proliferating population of murine splenocytes, wasdetermined for 4-aza-OdDHL, 4-methyl-4-aza-OdDHL and for the compoundN-(2-fluoro-4-aza-3-oxododecanoyl)-L-homoserine lactone(2-fluoro-4-aza-OdDHL).

Also determined for each of these compounds was the autoinducer activityas a measure of the ability of the compound to induce light productionin a specifically designed bacterial bioreporter system. Autoinducer(A.I.) activity was measured by the ability of the test N-acylhomoserine lactone compound (AHL) to induce bioluminescence in E. colicontaining a lux-based bioluminescence reporter plasmid. For detectionof a long chain AHL, such as OdDHL, E. coli JM109 harbouring thereporter plasmid pSB1075 was used. This reporter contains the P.aeruginosa IasR gene and IasI promoter fused to IuxCDABE from P.luminescens and preferentially responds to AHLs with acyl chains of10-14 carbons in length.

Bioassays were performed in 96 well microtitre plates. Briefly, 10 μl ofthe compound being assessed was placed into a microtitre well plate andserially diluted using LB broth to produce a concentration range from100 μM to 100 fM. Similar dilutions were performed on a synthetic OdDHLand used as a positive control. One hundred microlitres of an overnightbiosensor strain was added to each well and light emission was measuredfollowing 4 hours incubation at 37° C. Light production was assessed ascounts per second on Perkin Elmer TopCount apparatus calibrated tomeasure bioluminescence. Values are expressed as a percentage of thebioluminescence measured after exposure of the bioreporter to OdDHL forfour hours at 37° C. Logarithmic regression analysis of the range ofsynthetic OdDHL dilutions typically returned coefficient ofdetermination values above 0.96.

The IC₅₀ (μM) and A.I. activity (10 μM) values obtained for the testcompounds and for OdDHL are shown in the following Table.

IC₅₀* AI activity** Name Structure (μM) (10 μM) 4-aza-OdDHL 17.1 2.8%2-fluoro-4-aza-OdDHL 21.8 19.9% 4-methyl-4-aza-OdDHL 14.6 23.2% OdDHL12.7 100.0% *IC₅₀ = dose of compound required to inhibit 50% of aproliferating population of murine splenocytes. Measured using[³H]-thymidine incorporation as a marker of proliferation and determinedby nonlinear regressions analysis. All coefficient of determinationvalues were in excess of 0.98. **Autoinducer activity is a measure ofthe ability of the compound to induce light production in a specificallydesigned bacterial bioreporter system. Values are expressed as apercentage of the bioluminescence measured after exposure of thebacteria to OdDHL for 4 hours at 37° C.

1. A compound of the formula I,

wherein R¹ is a saturated or unsaturated straight chain or branchedchain aliphatic hydrocarbyl group containing from 5 to 14 carbon atoms;R² is H or a 1-4C alkyl group; R³ is H or F; and any enantiomer thereof.2. A compound according to claim 1, wherein R¹ is a straight chain alkylgroup containing from 5 to 14 carbon atoms.
 3. A compound according toclaim 2, wherein R¹ is a n-octyl group.
 4. A compound according to anyone of claims 1 to 3, wherein the group R² is selected from H or amethyl group.
 5. A compound according to claim 1 which isN-(4-aza-3-oxododecanoyl)-L-homoserine lactone.
 6. A compound accordingto claim 1 which is N-(4-methyl-4-aza-3-oxododecanoyl)-L-homoserinelactone.
 7. A compound according to claim 1 which isN-(4-aza-2-fluoro-3-oxododecanoyl)-L-homoserine lactone.
 8. Apharmaceutical composition comprising, as active component, a compoundaccording to any one of claims 1 to 7 and one or morepharmaceutically-acceptable carrier, excipient or diluent.
 9. A methodof making a compound of the formula I,

wherein R¹ is a saturated or unsaturated straight chain or branchedchain aliphatic hydrocarbyl group containing from 5 to 14 carbon atoms;R² is H or a 1-4C alkyl group; R³ is H or F; and any enantiomer thereof,which method comprises reacting a substituted carboxylic acid having theformula R¹R²NC(O)CHR³C(O)OH, where R¹, R² and R³ are defined above, withN,N′-carbonyldiimidazole and then reacting the N-acylimidazoleintermediate with L-homoserine lactone.